Methods and compositions for inhibition of the transitional endoplasmic reticulum ATPase

ABSTRACT

Compounds of Formulas I-XLIII are identified as direct inhibitors of p97 ATPase or of the degradation of a p97-dependent ubiquitin-proteasome system (UPS) substrate. Methods and compositions are disclosed for inhibiting p97 ATPase and the degradation of a p97-dependent UPS substrate, and for identifying inhibitors thereof.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims priority to and the benefit of U.S. Provisional Application Ser. No. 61/332,667, filed on May 7, 2010, the entire contents of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under MH085687 awarded by National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

Embodiments of this invention are directed to selective inhibitors of the ubiquitin-proteasome system (UPS). In particular, inhibitors of the transitional endoplasmic reticulum (p97) ATPase and an ubiquitin substrate are identified.

TECHNICAL BACKGROUND

The ubiquitin-proteasome system (UPS) comprises one of the most important mechanisms for post-translational regulation of protein function in eukaryotic cells. The UPS comprises hundreds of enzymes that promote covalent attachment of ubiquitin and ubiquitin-like proteins (UBL) to target proteins, as well as enzymes that reverse the modification. Conjugation of ubiquitin to target proteins is a multi-step process (Weissman, 2001, Nat. Rev. Mol. Cell. Biol., 2:169-178; Finley, 2009, Annu. Rev. Biochem., 78:477-513; Schrader et al., 2009, Nat. Chem. Biol., 5:815-822; Deshaies et al., 2009, Annu. Rev. Biochem., 78: 399-434). The most intensively-studied consequence of ubiquitination is protein degradation. Given the importance of the UPS to regulatory biology there has been considerable interest in developing small molecule inhibitors as potential therapies for a range of human diseases. The UPS has been validated as an important target in cancer by clinical use of the proteasome inhibitor, bortezomib (Velcade), for the treatment of multiple myeloma and mantle cell lymphoma (Kane et al., 2003, Oncologist, 8:508-513; Colson et al., 2004, Clin. J. Oncol. Nurs.,8:473-480).

The AAA (ATPase associated with diverse cellular activities) ATPase p97 is conserved across all eukaryotes and is essential for life in budding yeast (Giaever et al., 2002, Nature, 418:387-391) and mice (Muller et al., 2007, Biochem. Biophys. Res. Commun., 354:459-465). p97 (also referred to as the transitional endoplasmic reticulum ATPase) is overexpressed in several cancers supporting the idea that it could be a target of general importance in oncology (Yamamoto et al., 2004, Clin. Cancer Res., 10:5558-5565; Yamamoto et al., 2003, J. Clin. Oncol., 21:447-452). Elevated expression levels of p97 have been associated with poor prognosis of cancer (Yamamoto et al., 2004, Ann. Surg. Oncol. 11:697-704; Tsujimoto et al., 2004, Clin. Cancer Res., 10:23007-3012). Additionally, p97 is an essential ATP hydrolase and thus, it should be druggable and have antiproliferative activity. Furthermore, p97 is essential for endoplasmic reticulum associated degradation (ERAD) (Ye et al., 2004, Nature, 429:841-847; Ye et al., 2003, J. Cell Biol., 162:71-84; Neuber et al., 2005, Nat. Cell Biol., 7:993-998). Blockade of ERAD is thought to be a key mechanism underlying the anti-cancer effects of bortezomib (Nawrocki et al., 2005, Cancer Res., 65:11510-11519). Given that p97 is implicated in ERAD, but otherwise has a more restricted role in the UPS compared to the proteasome, it is possible that drugs that target p97 might retain much of the efficacy of bortezomib but with less toxicity.

SUMMARY

In one embodiment of the present invention, a method of decreasing p97 ATPase activity and/or degradation of a p97-dependent ubiquitin-proteasome system (UPS) substrate in a human cell, is provided, including administering to a human an effective amount of at least one of (i) a compound represented by any of Formulas I-VII, IX, XI-XLIII, (ii) a PEGylated analog of the compound, (iii) a pharmaceutically acceptable salt of said compound or analog, or (iv) an isomer of said compound, analog, or salt, wherein, for Formula I, R¹, R², R³, R⁴, and R⁵ are selected from the combinations listed in Tables 1.1, 12.1, and 18.1; wherein, for Formula II, R¹ is selected from the groups listed in Tables 2.1, 13.1, and 19.1; wherein, for Formula III, R¹ is selected from the groups listed in Table 3.1; wherein, for Formula IV, R¹ is selected from the groups listed in Table 4.1; wherein, for Formula V, R¹ is selected from the groups listed in Table 5.1; wherein, for Formula VI, R¹ is selected from the groups listed in Tables 6.1 and 20.1; wherein, for Formula VII, R¹, n, X, and Y are selected from the combinations listed in Tables 7.1 and 14.1; wherein for Formula XI, R² is selected from the groups listed in Tables 9.1 and 21; wherein for Formula XII, R⁴ is selected from the groups listed in Tables 10.1 and 22.1; wherein, for Formula XXI, R1 is 5,6-dimethyl; and wherein, for Formula XXV, R¹ is chlorine at position 3 and R² is selected from hydrogen and methoxy at position 4.

In one embodiment, the preceding method is provided wherein the compound decreases p97 ATPase activity and degradation of a p97-dependent UPS substrate in a human cell, and the compound is represented by any of Formulas I-VII, IX, and XI-XIX, wherein: for Formula I, R¹, R², R³, R⁴, and R⁵ are selected from the combinations listed in Table 1.1, for Formula II, R¹ is selected from the groups listed in Table 2.1, for Formula III, R¹ is selected from the groups listed in Table 3.1, for Formula IV, R¹ is selected from the groups listed in Table 4.1, for Formula V, R¹ is selected from the groups listed in Table 5.1, for Formula VI, R¹ is selected from the groups listed in Table 6.1, for Formula VII, R¹, n, X, and Y are selected from the combinations listed in Table 7.1, for Formula XI, R² is selected from the groups listed in Table 9.1, and for Formula XII, R⁴ is selected from the groups listed in Table 10.1.

In one embodiment, the preceding method is provided wherein the isomer is a regioisomer or a stereoisomer.

In one embodiment, a method of identifying an inhibitory compound that decreases p97 activity in a human cell is provided, including: (a) forming a p97 protein control solution; (b) forming a test solution comprising p97 protein and at least one of (i) a compound represented by any of Formulas LII through LXVI, (ii) a PEGylated, biotinylated, or fluorescently labeled analog of the compound, (iii) a pharmaceutically acceptable salt of said compound or analog, or (iv) an isomer of said compound analog, or salt; (c) measuring p97 activity of the control solution and of the test solution in the presence of ATP and a kinase; and (d) comparing the measured activities, wherein for Formula LII, n is selected from 0, 1, and 2; wherein, for Formula LIII, R¹ and R² are independently selected from hydrogen, methyl, ethyl, propyl and butyl; wherein, for Formula LIV, R¹ is selected from hydrogen, methyl, fluorine, chlorine, bromine, and OMe (methoxy), and R² is selected from hydrogen, methyl, ethyl, propyl, and butyl; wherein, for Formula LV, R¹ is selected from hydrogen, methyl, fluorine, chlorine, bromine, and OMe (methoxy), and R² is selected from hydrogen, methyl, ethyl, propyl, and butyl; wherein, for Formula LVI, R¹ and R² are independently selected from hydrogen, methyl, fluorine, chlorine, bromine, and OMe (methoxy); wherein, for Formula LVII, X is oxygen, NMe (nitrogen-methyl), NEt (nitrogen-ethyl), NPh (nitrogen-phenyl); n is selected from −1, 0, 1, and 2; m is selected from 1, 2, 3, and 4; and R¹ and R² are independently selected from hydrogen, methyl, fluorine, chlorine, bromine and methoxy; wherein, for Formula LVIII, X is selected from oxygen, NMe (nitrogen-methyl), NEt (nitrogen-ethyl), and NPh (nitrogen-phenyl); n is selected from −1, 0, 1, and 2; m is selected from 1, 2, 3, and 4; and R¹ is selected from hydrogen, methyl, fluorine, chlorine, bromine, and methoxy; wherein, for Formula LIX, R¹, R² and R³ are independently selected from hydrogen, A(CH₂)_(n)CH₃, and A(CH₂)_(n)X, where n is selected from 0, 1, 2, 3, 4 and 5, A is O, S or NH and X is selected from heteroaryl, O(alkyl), S(alkyl), (O-alkyl)₂, and (S-alkyl)₂; wherein, for Formula LX, A¹ is selected from O, S, Se, N, NH, CH, CH₂, CHalkyl, and Calkyl, wherein n is 1 or 2; A² is selected from N, NH, CH, and Calkyl; and R¹ is selected from H, A(CH₂)_(n)CH₃, or A(CH₂)_(n)X, where A is O, S or NH and X is heteroaryl, O(alkyl), S(alkyl), (O-alkyl)₂, or (S-alkyl)₂; and n is 0, 1, 2, 3, 4, or 5; wherein, for Formula LXI, R¹, R² and R³ are independently selected from alkyl, alkoxyalkyl, and aminoalkyl; wherein, for Formula LXII; n is selected from −1, 0, 1, and 2; m is selected from 0, 1, and 2; and X is selected from CH₂, O, NMe, NEt, and NPh; wherein, for Formula LXIII, R¹ and R² are independently selected from H, Me, Et, Pr, and Bu; X is selected from CH₂, O, NMe, NEt, and NPh; and n is selected from −1, 0, 1 and 2; wherein, for Formula LXIV, R¹ is selected from H, Me, F, Cl, Br, and OMe; R² is selected from H, Me, Et, Pr, and Bu; X is selected from CH₂, O, NMe, NEt, and NPh; and n is selected from −1, 0, 1, and 2; wherein, for Formula LXV, R¹ is selected from H, Me, F, Cl, Br, and OMe; R² is selected from H, Me, Et, Pr, and Bu; X is selected from CH₂, O, NMe, NEt, and NPh; and n is selected from −1, 0, 1, and 2; and, wherein, for Formula LXVI, R¹ and R² are independently selected from H, Me, F, Cl, Br, and OMe; X is selected from CH2, O, NMe, NEt, and NPh; and n is selected from −1, 0, 1, and 2.

In one embodiment, the preceding composition is provided wherein the isomer is a regioisomer or a stereoisomer.

In one embodiment, a composition for decreasing p97 ATPase activity and/or degradation of a p97-dependent UPS substrate is provided, including: at least one of (i) a compound selected from any of Formulas II-VII, IX, XI, XII, XX, XXI, XXIV, XXV, XLII, and XLIII, (ii) a PEGylated, biotinylated, or fluorescently labeled analog of the compound, (iii) a pharmaceutically acceptable salt of said compound or analog; or (iv) an isomer of said compound analog, or salt, wherein, for Formula II, R¹ is selected from the groups listed in Tables 2.2, 13.1, and 19.1; wherein, for Formula III, R¹ is selected from the groups listed in Table 3.1; wherein, for Formula IV, R¹ is selected from the groups listed in Table 4.1; wherein, for Formula V, R¹ is selected from the groups listed in Table 5.1; wherein, for Formula VI, R¹ is selected from the groups listed in Tables 6.1 and 20.1; wherein, for Formula VII, R′, n, X, and Y are selected from the combinations listed in Tables 7.1 and 14.1; wherein, for Formula XI, R² is selected from the groups listed in Tables 9.1 and 21; wherein, for Formula XII, R⁴ is selected from the groups listed in Tables 10.1 and 22.1; and wherein, for Formula XXV, R¹ is chlorine at position 3 and R² is selected from hydrogen and methoxy at position 4.

In one embodiment, the preceding composition for decreasing p97 ATPase activity and/or degradation of a p97-dependent UPS substrate is provided further including a pharmaceutically acceptable carrier.

In one embodiment, the preceding composition for decreasing p97 ATPase activity and/or degradation of a p97-dependent UPS substrate is provided, wherein the isomer is a regioisomer or a stereoisomer.

In one embodiment, the preceding composition decreases p97 ATPase activity and degradation of a p97-dependent UPS substrate, and the compound is represented by any of Formulas II-VII, IX, XI, and XII, wherein, for Formula II, R¹ is selected from the groups listed in Table 2.2; for Formula III, R¹ is selected from the groups listed in Table 3.1; for Formula IV, R¹ is selected from the groups listed in Table 4.1; for Formula V, R¹ is selected from the groups listed in Table 5.1; for Formula VI, R¹ is selected from the groups listed in Table 6.1; for Formula VII, R¹, n, X, and Y are selected from the combinations listed in Table 7.1; for Formula XI, R² is selected from the groups listed in Table 9.1; and for Formula XII, R⁴ is selected from the groups listed in Table 10.1.

In one embodiment, the preceding composition that decreases p97 ATPase activity and degradation of a p97-dependent UPS substrate is provided, further including a pharmaceutically acceptable carrier.

In one embodiment, the preceding composition that decreases p97 ATPase activity and degradation of a p97-dependent UPS substrate is provided, wherein the isomer is a regeoisomer or a stereoisomer.

In one embodiment, a composition for identifying an inhibitor that decreases p97 ATPase activity and/or degradation of a p97-dependent UPS substrate is provided, including: at least one of (i) a compound selected from any of Formulas LII-LXVI, (ii) a PEGylated, biotinylated, or fluorescently labeled analog of the compound, (iii) a pharmaceutically acceptable salt of said compound or analog; or (iv) a isomer of said compound, analog, or salt: wherein for Formula LII, n is selected from 0, 1, and 2; wherein, for Formula LIII, R¹ and R² are independently selected from hydrogen, methyl, ethyl, propyl and butyl; wherein, for Formula LIV, R¹ is selected from hydrogen, methyl, fluorine, chlorine, bromine, and OMe (methoxy), and R² is selected from hydrogen, methyl, ethyl, propyl, and butyl; wherein, for Formula LV, R¹ is selected from hydrogen, methyl, fluorine, chlorine, bromine, and OMe (methoxy), and R² is selected from hydrogen, methyl, ethyl, propyl, and butyl; wherein, for Formula LVI, R¹ and R² are independently selected from hydrogen, methyl, fluorine, chlorine, bromine, and OMe (methoxy); wherein, for Formula LVII, X is oxygen, NMe (nitrogen-methyl), NEt (nitrogen-ethyl), NPh (nitrogen-phenyl); n is selected from −1, 0, 1, and 2; m is selected from 1, 2, 3, and 4; and R¹ and R² are independently selected from hydrogen, methyl, fluorine, chlorine, bromine and methoxy; wherein, for Formula LVIII, X is selected from oxygen, NMe (nitrogen-methyl), NEt (nitrogen-ethyl), and NPh (nitrogen-phenyl); n is selected from −1, 0, 1, and 2; m is selected from 1, 2, 3, and 4; and R¹ is selected from hydrogen, methyl, fluorine, chlorine, bromine, and methoxy; wherein, for Formula LIX, R¹, R² and R³ are independently selected from hydrogen, A(CH₂)_(n)CH₃, and A(CH₂)_(n)X, where n is selected from 0, 1, 2, 3, 4 and 5, A is O, S or NH and X is selected from heteroaryl, O(alkyl), S(alkyl), (O-alkyl)₂, and (S-alkyl)₂; wherein, for Formula LX, A¹ is selected from O, S, Se, N, NH, CH, CH₂, CHalkyl, and Calkyl, wherein n is 1 or 2; A² is selected from N, NH, CH, and Calkyl; and R¹ is selected from H, A(CH₂)_(n)CH₃, or A(CH₂)_(n)X, where A is O, S or NH and X is heteroaryl, O(alkyl), S(alkyl), (O-alkyl)₂, or (S-alkyl)₂; and n is 0, 1, 2, 3, 4, or 5; wherein, for Formula LXI, R¹, R² and R³ are independently selected from alkyl, alkoxyalkyl, and aminoalkyl; wherein, for Formula LXII; n is selected from −1, 0, 1, and 2; m is selected from 0, 1, and 2; and X is selected from CH₂, O, NMe, NEt, and NPh; wherein, for Formula LXIII, R¹ and R² are independently selected from H, Me, Et, Pr, and Bu; X is selected from CH₂, O, NMe, NEt, and NPh; and n is selected from −1, 0, 1 and 2; wherein, for Formula LXIV, R¹ is selected from H, Me, F, Cl, Br, and OMe; R² is selected from H, Me, Et, Pr, and Bu; X is selected from CH₂, O, NMe, NEt, and NPh; and n is selected from −1, 0, 1, and 2; wherein, for Formula LXV, R¹ is selected from H, Me, F, Cl, Br, and OMe; R² is selected from H, Me, Et, Pr, and Bu; X is selected from CH₂, O, NMe, NEt, and NPh; and n is selected from −1, 0, 1, and 2; and, wherein, for Formula LXVI, R¹ and R² are independently selected from H, Me, F, Cl, Br, and OMe; X is selected from CH₂, O, NMe, NEt, and NPh; and n is selected from −1, 0, 1, and 2.

In one embodiment, the preceding composition for identifying an inhibitor is provided, further including a pharmaceutically acceptable carrier.

In one embodiment, the preceding composition for identifying an inhibitor is provided, wherein the isomer is a regeoisomer or a stereoisomer.

In one embodiment, a method of decreasing p97 ATPase activity and/or degradation of a p97-dependent ubiquitin-proteasome system (UPS) substrate in a human cell is provided, including: administering to a human an effective amount of at least one of (i) a compound represented by any of Formulas VIII, X, XI, and XII, (ii) a PEGylated analog of the compound, (iii) a pharmaceutically acceptable salt of said compound or analog; or (iv) an isomer of said compound, analog, or salt, wherein, for Formula XI, R² is selected from the groups listed in Tables 9.2 and 15.1; and wherein, for Formula XII, R⁴ is selected from the groups listed in Tables 10.2 and 22.2.

In one embodiment, a composition for decreasing p97 ATPase activity and/or degradation of a p97-dependent UPS substrate is provided, including at least one of (i) a compound selected from any of Formulas VIII, X, XI, XII, (ii) a PEGylated, biotinylated, or fluorescently labeled analog of the compound, (iii) a pharmaceutically acceptable salt of said compound, or (iv) an isomer of said compound, analog, or salt, wherein, for Formula XI, R² is selected from the groups listed in Tables 9.2 and 15.1, and wherein, for Formula XII, R⁴ is selected from the groups listed in Tables 10.2 and 22.2.

In one embodiment, the preceding composition for decreasing p97 ATPase activity and/or degradation of a p97-dependent UPS substrate is provided, further including a pharmaceutically acceptable carrier.

In one embodiment, the preceding composition for decreasing p97 ATPase activity and/or degradation of a p97-dependent UPS substrate is provided, wherein the isomer is a regeoisomer or a stereoisomer.

DETAILED DESCRIPTION

Utilizing a set of dual-reporter human cell lines and a chase protocol to quantify and distinguish p97-specific inhibitors of proteasomal degradation, compounds that directly inhibit p97 and inhibit the degradation of a UPS substrate that depends on p97 were identified and characterized.

Compounds were identified as “inhibitors” if the compound had an IC₅₀ of 20 μM or less (potency). Inhibition was measured using a p97 ATPase assay and a p97-dependent Ub^(G76V)-GFP assay that measures Ub^(G76V)-GFP turn over. In one embodiment, a compound of the present invention decreases p97 activity and/or p97-dependent Ub^(G76V)-GFPdegradation turn-over to 20 μM or less. In another embodiment, the compound decreases the p97 ATPase activity and/or p97-dependent Ub^(G76V)-GFPdegradation turn-over to 15 μM or less. In another embodiment, the compound decreases the p97 ATPase activity and/or p97-dependent Ub^(G76V)-GFP degradation turn-over to 10 μM or less. In a preferred embodiment, the compound decreases the p97 ATPase activity and/or p97-dependent Ub^(G76V)-GFP degradation turn-over is decreased to 5 μM or less. In a most preferred embodiment, the compound decreases the p97 ATPase activity and/or p97-dependent Ub^(G76V)-GFP degradation turn-over to 2 μM or less.

Compounds were categorized into three types of inhibitors: 1) inhibitors of both p97 and Ub^(G76V)-GFP turn-over (degradation); 2) inhibitors of p97 that do not inhibit Ub^(G76V)-GFP turn over; and 3) inhibitors of Ub^(G76V)-GFP turn-over that do not inhibit p97. Comparative Examples are shown in Tables 28-33, listing compounds assayed that did not decrease either p97 or Ub^(G76V)-GFP turnover to at least 20 μM.

In one embodiment, a method for decreasing p97 ATPase activity and/or decreasing the degradation of the p97-dependent UPS substrate (Ub^(G76V)-GFP), is carried out using a compound represented by one of Formulas I-XLIII and, where applicable, having variable groups as shown in Tables 1-26.

As shown in the tables throughout, compounds and variable groups are characterized using abbreviations, which are defined as follows. In the * column of the tables, “P” refers to a purchased compound and “S” refers to a synthesized compound. R groups include H (hydrogen), C (carbon) N (nitrogen) Cl (chlorine), F (flourine), Br (bromine), NO₂ (nitro), Me (methyl), OMe (methoxy), Ph (phenyl), PhOMe (methoxyphenyl). Standard IUPAC nomenclature is followed for all chemical abbreviations unless indicated otherwise. Numbers preceding the atom groups indicate the position for that atom. Specific compound data is found in the enclosed NMR data (Example 9, Appendix).

Inhibitors of p97 and p97-Dependent UPS Substrate (Ub^(G76V)-GFP)

Tables 1-11 show compounds represented by Formulas I-XIX having an IC₅₀ of 20 μM or less in both a p97 ATPase assay (Example 7) and a p97-dependent Ub^(G76V)-GFP degradation turn-over assay (Example 8).

Formula I

TABLE 1 FORMULA I:

IC₅₀ (μM) KU Ub^(G76V)-GFP Cpd CID SID SCC # * R1 R2 R3 R4 R5 ATPase turn over I-1 8868 87796 KSC-1- P H 2-F H H H   3 ± 0.8 9.0 ± 1.3 13 231 150 I-1 8868 96022 KSC-16- S H 2-F H H H 1.1 ± 0.3   8 ± 1.7 13 089 88 I-2 7806 87796 KSC-1- P H H H H H 4.2 ± 2.3 12 ± 3  43 227 145 I-3 1894 87796 KSC-1- P H 2-Cl H H H 1.2 ± 0.6 8 ± 3 007 228 146 I-4 2927 87796 KSC-1- P H 3-NO₂ H H H 5.9 ± 3   4.0 ± 1.6 831 230 149 I-5 4084 87796 KSC-1- P H 3-Me H H H 4.2 ± 0.2 17 ± 4  712 265 226 I-6 1591 87796 KSC-1- P H 4-Cl H H H 5.5 ± 1.9 20 ± 3  117 273 236 I-8 1187 87796 KSC-1- P H 4-NO₂ H H H 11 ± 6  19 ± 7  251 271 234 I-9 9497 87796 KSC-1- P H 4-OMe H H H 1.6 ± 0.3 15 ± 2  42 266 227 I-10 3395 87796 KSC-1- P H 4-Me H H H 5.7 ± 0.7 13 ± 2  671 263 224 I-13 2452 87796 KSC-1- P H H H H 4-F 11 ± 3  19 ± 3  802 251 212 I-17 2096 87796 KSC-1- P H 4-OMe H H 4-F 7.8 ± 0.4 9.8 ± 3   3125 258 219 I-20 2096 87796 KSC-1- P H 4-OMe H H 4- 13 ± 4  15 ± 5  3158 259 220 OMe I-21 2096 87796 KSC-1- P H 4-OMe H H 4-Me 8 ± 2 12 ± 2  3177 260 221 I-22 2096 87796 KSC-1- P H 4-OMe H H 4-Cl 8 ± 2 9.3 ± 1   3187 261 222 I-23 2096 87796 KSC-1- P H 4-Cl H H 4-F   8 ± 0.2 20 ± 5  3255 262 223 I-24 4084 87796 KSC-1- P H 3-Me H H 4-F 15 ± 2   12 ± 0.8 711 264 225 I-30 2790 87796 KSC-1- P H 4-Me H Me H 7 ± 4 17 ± 4  952 274 237

Formula II

TABLE 2 FORMULA II:

IC₅₀ (μM) Ub^(G76V)- KU GFP Cpd # CID SID SCC # * R1 ATPase turn over II-1 2909 87796 KSC-1- P H 2.3 ± 1   3.1 ± 0.4 934 234 153 II-2 9295 87796 KSC-1- P 4-Me 2.2 ± 0.4 6.3 ± 1.4 48 285 251 II-3 2351 87796 KSC-1- P 4-Cl 5.4 ± 2   5.9 ± 1.2 737 281 246 II-4 7976 87796 KSC-1- P 4-F 3.8 ± 1.2 9.4 ± 1   50 284 249 II-5 1943 87796 KSC-1- P 4-Br 1.8 ± 0.4 6.0 ± 0.8 389 280 245 II-6 1330 92093 KSC-1- P 4-OMe 3.8 ± 0.5 4.5 ± 1.1 474 141 250 II-6 1330 92252 KSC-1- S 4-OMe 3.5 ± 0.5 4.8 ± 0.7 474 642 290 II-7 9494 87796 KSC-1- P 2-Me 3.4 ± 1.5 10 ± 2  45 286 252 II-9 8861 87796 KSC-1- P 2-F 0.85 ± 0.17 10 ± 2  96 282 247 II-11 1415 87796 KSC-1- P 2-OMe 2.2 ± 0.9 11 ± 3  819 283 248 II-12 9500 87796 KSC-1- P 3-Me 1.7 ± 0.5 3.0 ± 0.7 33 287 253 II-13 1633 87796 KSC-1- P 3-Cl 0.48 ± 0.16 7.8 ± 1.3 082 279 244 II-14 1164 92252 KSC-1- S 3-F 1.6 ± 0.1 2.7 ± 0.4 5888 644 292 II-15 4510 92252 KSC-1- S 3-Br 2.5 ± 0.5 4.3 ± 0.8 8365 645 293 II-16 3986 92252 KSC-1- S 3-OMe 2.5 ± 0.2 4.3 ± 0.7 1404 643 291 II-17 1571 87796 KSC-1- P 3,4-di-Cl 8.1 ± 2.6 12 ± 2  079 290 259 II-18 4510 92252 KSC-1- S 3-Cl-6-F 13 ± 3  13 ± 2  8364 647 295 II-19 4510 92252 KSC-1- S 3,5-di-Cl 8.2 ± 0.3 13 ± 1  8364 647 294 II-22 4685 99239 KSC-16- S 3-NO₂ 5.2 ± 0.5 19 ± 2  0871 933 155

Formula III

TABLE 3 FORMULA III:

IC₅₀ (μM) Ub^(G76V)-GFP Cpd # CID SID KU SCC # * R1 ATPase turn over III-1 4617 9602 KSC-16-72 S 4-Me 1.4 ± 0.3  12 ± 2 3070 2083 III-2 4617 9602 KSC-16-89 S 4-Cl 2.8 ± 0.9 7.6 ± 2.4 3066 2090 III-3 4617 9602 KSC-16-98 S 4-F 1.5 ± 0.3 5.7 ± 1.3 3057 2093 III-4 4617 9602 KSC-16-101 S 4-Br 7.4 ± 2  10 ± 3 3059 2096 III-5 4617 9602 KSC-16-79 S 4-OMe 2.3 ± 0.6  11 ± 4 3063 2086 III-6 4617 9602 KSC-16-63 S 2-Me 4.2 ± 1.1  11 ± 3 3069 2080 III-7 4617 9602 KSC-16-84 S 2-Cl 4.7 ± 1.4  18 ± 5 3062 2087 III-8 4617 9602 KSC-16-92 S 2-F 1.8 ± 0.5 7.8 ± 2.1 3072 2091 III-9 4617 9602 KSC-16-99 S 2-Br   4 ± 1.7  12 ± 5 3058 2094 III-10 4617 9602 KSC-16-75 S 2-OMe 1.6 ± 0.4  11 ± 2 3067 2084 III-11 4617 9602 KSC-16-66 S 3-Me 2.3 ± 0.6 9.7 ± 2.7 3061 2081 III-12 4617 9602 KSC-16-87 S 3-Cl   5 ± 1.2  15 ± 4 3068 2088 III-13 4617 9602 KSC-16-95 S 3-F 1.2 ± 0.2 6.3 ± 1.7 3060 2092 III-14 4617 9602 KSC-16-100 S 3-Br 3.7 ± 0.9 9.9 ± 2 3065 2095 III-15 4617 9602 KSC-16-78 S 3-OMe   1 ± 0.1 9.9 ± 1.6 3071 2085

Formula IV

TABLE 4 FORMULA IV:

IC₅₀ (μM) Ub^(G76V)-GFP Cpd CID SID KU SCC # * R1 ATPase turn over IV-1 2514 9337 KSC-1-300 S 4-Me   3 ± 0.3 1.5 ± 0.4 4450 4186 IV-2 4538 9337 KSC-16-33 S 4-Cl 3.1 ± 0.2 2.1 ± 0.4 2112 4224 IV-3 4538 9337 KSC-16-38 S 4-F 2.6 ± 0.2 3.4 ± 0.5 2121 4227 IV-4 4538 9337 KSC-16-42 S 4-Br 4.3 ± 0.9 3.9 ± 0.3 2119 4230 IV-5 4538 9337 KSC-16-2 S 4-OMe   3 ± 0.5 1.3 ± 0.2 2111 4187 IV-6 4538 9337 KSC-16-8 S 2-Me 2.7 ± 0.5 1.9 ± 0.4 2120 4191 IV-7 4538 9337 KSC-16-31 S 2-Cl 5.3 ± 1.2 2.9 ± 0.9 2117 4222 IV-8 4538 9337 KSC-16-35 S 2-F 2.9 ± 0.4 2.8 ± 0.4 2108 4225 IV-9 4538 9337 KSC-16-40 S 2-Br 2.0 ± 0.3 3.8 ± 0.7 2107 4228 IV-10 4538 9337 KSC-16-29 S 2-OMe 3.6 ± 0.8 1.9 ± 0.2 2105 4193 IV-11 4538 9337 KSC-16-28 S 3-Me 2.3 ± 0.4 2.5 ± 0.5 2113 4192 IV-12 4538 9337 KSC-16-32 S 3-Cl 2.7 ± 0.1 2.5 ± 0.4 2114 4223 IV-13 4538 9337 KSC-16-36 S 3-F 3.1 ± 0.4 2.8 ± 0.4 2110 4226 IV-14 4538 9337 KSC-16-41 S 3-Br 3.7 ± 0.4 2.8 ± 0.4 2118 4229 IV-15 4538 9337 KSC-16-30 S 3-OMe 4.5 ± 0.8 2.5 ± 0.8 2116 4221 IV-16 4538 9337 KSC-16-6 S 4-CF₃ 2.6 ± 0.4 2.3 ± 0.3 2109 4190 IV-17 4538 9337 KSC-16-3 S 3,4-di-Cl   3 ± 0.5 1.9 ± 0.2 2106 4188 IV-18 4538 9337 KSC-16-4 S 4-Cl-3-CF₃ 4.7 ± 1 2.2 ± 0.4 2115 4189

Formula V

TABLE 5 FORMULA V:

IC₅₀ (μM) Ub^(G76V)- KU SCC GFP Cpd CID SID # * R1 ATPase turn over V-1 4622 9607 KSC-16- S 3-Cl-2-OMe 0.9 ± 0.2 6.3 ± 1.7 4527 9523 103 V-2 4622 9607 KSC-16- S 3-F-2-Me 0.9 ± 0.1 3.7 ± 0.8 4522 9524 104 V-3 4622 9607 KSC-16- S 3-F-5-Me 0.7 ± 0.05 5.4 ± 0.8 4524 9525 105 V-4 4622 9607 KSC-16- S 3-F-2-OMe 1.7 ± 0.5  12 ± 3 4529 9526 105 V-5 4622 9607 KSC-16- S 3-Cl-2-Me 0.7 ± 0.1 5.5 ± 1 4523 9527 107 V-6 4622 9607 KSC-16- S 3-F-6-Me 0.8 ± 0.1   5 ± 1 4519 9528 108 V-7 4622 9607 KSC-16- S 3-F-4-Me 0.9 ± 0.2 5.2 ± 1.2 4528 9529 109 V-8 4622 9607 KSC-16- S 3-F-4-OMe 0.9 ± 0.1 4.7 ± 0.8 4530 9530 110 V-9 4622 9607 KSC-16- S 3-Cl-6-Me 0.8 ± 0.07   7 ± 1.5 4526 9531 112 V-10 4622 9607 KSC-16- S 3-Cl-6-OMe 1.1 ± 0.2  16 ± 6 4518 9532 113 V-11 4682 9920 KSC-16- S 3-F-6-OMe 6.7 ± 3 9.3 ± 1 9342 6553 120

Formula VI

TABLE 6 FORMULA VI:

IC₅₀ (μM) Ub^(G76V)- Cpd KU SCC GFP # CID SID # * R1 ATPase turn over VI-1 4622 9607 KSC-16- S 5-Cl 2.2 ± 0.7  16 ± 2 4525 9533 114 VI-2 4622 9607 KSC-16- S 5-F 1.2 ± 0.3  13 ± 2 4521 9534 115 VI-3 4622 9607 KSC-16- S 6-Cl 1.6 ± 0.4 6.6 ± 0.9 4520 9535 117 VI-4 5276 9602 KSC-16- S 6,7-di-OMe 4.4 ± 1.8 8.8 ± 1.5  745 2097 102 VI-5 4682 9920 KSC-16- S 8-OMe 0.6 ± 0.06  10 ± 2 9340 6522 121 VI-6 4682 9920 KSC-16- S 7-Me 2.2 ± 0.4 6.1 ± 1.6 9333 6552 118 VI-7 4685 9923 KSC-16- S 7-CF₃ 9.1 ± 2  19 ± 1 0874 9928 160 V1-9 4685 9923 KSC-16- S 8-Br 3.3 ± 0.9  30 ± 2 0882 9931 153 VI-10 4685 9923 KSC-16- S 8-F 3.1 ± 0.5  18 ± 2 0883 9932 154 VI-11 4685 9923 KSC-16- S 7-F 2.6 ± 0.5 6.1 ± 0.8 0884 9934 156 VI-12 4685 9923 KSC-16- S 7-Cl 5.5 ± 0.9 6.3 ± 0.9 0880 9935 159 VI-13 4685 9923 KSC-16- S 7-OMe 5.7 ± 1  42 ± 6 0885 9938 166 VI-14 4685 9923 KSC-16- S 6-OMe 7.5 ± 1.1 8.6 ± 1.7 0877 9939 172 VI-15 4685 9923 KSC-16- S 6-F 4.7 ± 0.4 8.2 ± 1.5 0873 9941 175

Formula VII

TABLE 7 FORMULA VII:

IC₅₀ (μM) Ub^(G76V)-GFP Cpd # CID SID KU SCC # * R1 n X Y** ATPase turn over VII-1 4682 9923 KSC-16-125 S H 1 CH₂ H, H  4.4 ± 1.8  10 ± 2 9336 9922 VII-2 4682 9923 KSC-16-144 S H 0 CH₂ H, H   2 ± 0.5  11 ± 3 9332 9923 VII-3 4617 9602 KSC-16-70 S H 1 O H, H  0.6 ± 0.2   7 ± 1.9 3064 2082 VII-4 4682 9920 KSC-16-147 S H 1 NH H, H  0.4 ± 0.05 6.3 ± 1 9338 6557 VII-5 4687 9931 KSC-16-182 S 8-OMe 1 NH H, H  0.9 ± 0.1 3.7 ± 0.2 3816 3586 VII-6 4687 9931 KSC-16-191 S 8-OMe 1 O H, H  0.2 ± 0.02 3.3 ± 0.4 3819 3591 VII-7 4693 9943 KSC-16-232 S 8-OH 1 O H, H  1.2 ± 0.3 7.7 ± 1.7 1212 7738 VII-8 4983 1039 KSC-16-255 S 8-Ph 1 O H, H  3.5 ± 0.6   5 ± 0.9 0264 0418   0 VII-9 4983 1039 KSC-16-260 S 8-OCH₂CH₂OH 1 O H, H 0.17 ± 0.05 3.8 ± 0.8 0253 0418   1 VII-10 4983 1039 KSC-16-265 S 8OCH₂CH₂NEt₂ 1 O H, H  0.4 ± 0.08 5.3 ± 0.6 0265 0418   3 VII-11 4983 1039 KSC-16-268 S 8-p-OMePh 1 O H, H  1.1 ± 0.1   9 ± 1 0267 0418   4 VII-12 4985 1042 KSC-25-17 S 8-OMe 1 NMe H, H  3.1 ± 0.5 7.8 ± 0.7 2177 2195   2 VII-13 4985 1042 KSC-25-15c1 S 8-OMe 1 NCOMe H, H  2.7 ± 0.6   8 ± 1 2173 2195   3 VII-14 4985 1042 KSC-25-29 S 8-OCH₂CH₂OMe 1 O H, H  0.6 ± 0.03 6.5 ± 0.7 2181 2195   7 VII-16 4983 1039 KSC-16-270 S 8-OMe 0 NH NH 0.11 ± 0.03 0.9 ± 0.1 0258 0416   9 VII-17 4983 1039 KSC-16-262cc S 8-OMe 0 O O 0.11 ± 0.03   5 ± 1 0270 0417   2

Formulas VIII to X

TABLE 8 IC₅₀ (μM) Cpd CID SID KU SCC # * ATPase Ub^(G76V)-GFP turn over VIII 4983 0260 1039 0418   5 KSC-16-290 S

Formula VIII 0.11 ± 0.03 3.5 ± 0.4 IX 4985 2184 1042 2195   0 KSC-25-14 S

Formula IX  0.4 ± 0.1 6.4 ± 1 X 4985 2172 1042 2195   5 KSC-25-24 S

Formula X  1.1 ± 0.2  10 ± 1

Formula XI

TABLE 9 Formula XI:

IC₅₀ (uM) Ub^(G76V)-GFP Cpd # CID SID KU SCC # * R2 ATPase turn over XI-5 4985 2185 10422  1947 KSC-25-22 S

 0.5 ± 0.2  14 ± 2 XI-6 4985 2183 10422  1949 KSC-25-21 S

 0.3 ± 0.07 7.8 ± 1.2 XI-8 2514 4452 99313  587 KSC-16-187 S

  15 ± 3   7 ± 1.3 XI-10 4693 1213 99437  735 KSC-16-203 S

 2.6 ± 0.4 2.8 ± 0.5 XI-11 4983 0269 10390  4171 KSC-16-273 S

  12 ± 5 2.1 ± 0.7 XI-13 4983 0255 10390  4174 KSC-16-278 S

 5.4 ± 0.9 4.2 ± 1 XI-14 4983 0257 10390  4175 KSC-16-282 S

 1.7 ± 0.4 1.8 ± 0.3 XI-15 4983 0266 10390  4176 KSC-16-283 S

  11 ± 1  10 ± 3 XI-16 4687 3815 99313  592 KSC-16-193 S

 0.5 ± 0.1 5.4 ± 1 XI-17 4687 3818 99313  593 KSC-16-194 S

0.52 ± 0.04 4.8 ± 0.7 XI-18 4687 3813 99313  594 KSC-16-196 S

 1.5 ± 0.2 7.1 ± 0.5

Formula XII

TABLE 10 FORMULA XII:

IC₅₀ (uM) Ub^(G76V)-GFP Cpd # CID SID KU SCC # * R4 ATPase turn over XII-4 49830  256 10390  4182 KSC-16-261 S

1.9 ± 0.4 3.7 ± 0.6 XII-5 49830  261 10390  4186 KSC-16-295 S

1.2 ± 0.2   3 ± 0.6 XII-6 49830  262 10390  4187 KSC-16-299 S

7.4 ± 0.9 8.2 ± 1 XII-7 46931  214 99437  736 KSC-16-219 S

 19 ± 5  15 ± 4 XII-8 46931  210 99437  737 KSC-16-222 S

4.6 ± 1   6 ± 0.6 XII-10 46931  211 99437  740 KSC-16-235c2 S

2.4 ± 0.5 7.3 ± 1

Formulas XIII-XIX

TABLE 11 IC₅₀ (uM) Cpd # KU SCC # * ATPase Ub^(G76V)-GFP turn over XIII KSC-16-13 P

Formula XIII 13 ± 5  10 ± 2 XIV KSC-16-16 P

Formula XIV 15 ± 5  14 ± 2.4 XV KSC-16-22 P

Formula XV 14 ± 5 3.7 ± 0.6 XVI KSC-16-23 P

Formula XVI 18 ± 8 5.9 ± 1 XVII KSC-16-24 P

Formula XVII 15 ± 4 5.7 ± 0.8 XVIII KSC-16-55 P

Formula XVIII 10 ± 4 6.3 ± 0.6 XIX KSC-16-56 P

Formula XIX 14 ± 6 7.2 ± 1.1

Inhibitors of p97

Tables 12-17 disclosed the compounds having an IC₅₀ of 20 μM or less in the p97 ATPase assay (Example 7), but did not decrease the p97-dependent Ub^(G76V)-GFP degradation turn-over assay to 20 μM or less (Example 8).

Formula I

TABLE 12 FORMULA I:

IC₅₀ (μM) Ub^(G76V)-GFP Cpd # CID SID KU SCC # * R1 R2 R3 R4 R5 ATPase turn over I-11 18190 8779 KSC-1-200 P H 3,4-di-Cl H H H 7.1 ± 0.4 37 ± 6   25 6240 I-28 15661 8779 KSC-1-235 P H 4-NO₂ Me H H 8.4 ± 4 28 ± 4   44 6272

Formula II

TABLE 13 Formula II:

IC₅₀ (μM) Ub^(G76V)-GFP Cpd CID SID KU SCC # * R1 ATPase turn over II-21 4685 9923 KSC-16-152 S 3-I 5.2 ± 1.9 36 ± 4 0881 9930

Formula VII

TABLE 14 Formula VII:

IC₅₀ (μM) Ub^(G76V)-GFP Cpd # CID SID KU SCC # * R1 n X Y ATPase turn over VII-15 4985 1042 KSC-25-30 S 8-nButyl 1 O H, H 2.63 ± 0.7 28 ± 3 2171 2195   8

Formula XI

TABLE 15 Formula XI:

IC₅₀ (μM) Ub^(G76V)-GFP Cpd # CID SID KU SCC # * R2 ATPase turn over XI-1 49852  176 10422  1943 KSC-25-3 S

3.3 ± 1.1     78 ± 45 XI-12 49830  259 10390  4173 KSC-16-277 S

  7 ± 3 >>20

Formula XX

TABLE 16 IC₅₀ (μM) Cpd # CID SID KU SCC # * ATPase Ub^(G76V)-GFP turn over XX 4985 2180 10422  1956 KSC-25-28 S

Formula XX 2.9 ± 0.2 27 ± 3

Formula XXI

TABLE 17 Formula XXI

IC₅₀ (μM) Ub^(G76V)-GFP Cpd CID SID KU SCC # * R1 ATPase turn over XXI-1 4985 10422 KSC-25-23 S 5,6-di-Me 2 ± 0.4 89 ± 39 2182  1948

Inhibitors of p97-Dependent UPS Substrate Ub^(G76V)-GFP

Tables 18-26 disclosed the compounds having an IC₅₀ of 20 μM or less in the p97-dependent Ub^(G76V)GFP degradation turn-over assay (Example 7), but did not decrease p97 to less than 20 uM.

Formula I

TABLE 18 FORMULA I:

IC₅₀ (μM) Ub^(G76V)-GFP Cpd CID SID KU SCC # * R1 R2 R3 R4 R5 ATPase turn over I-12  24527 8779 KSC-1-211 P H H H H 3-Cl 28 ± 9  17 ± 4 92 6250 I-14  24650 8779 KSC-1-214 P H H H H 2-Cl 30 ± 10 11 ± 3 33 6253 I-15  24563 8779 KSC-1-215 P H H H H 4-Cl >30 15 ± 3 09 6254 I-16 15993 8779 KSC-1-217 P H 2-F H H 2-Cl 26 ± 12 15 ± 3 188 6256 I-25  15538 8779 KSC-1-258 P H H Me H H 49 ± 17 17 ± 5 19 6289 I-26 15995 8779 KSC-1-218 P H 2-F Me H H 25 ± 4   6.0 ± 2.0 431 6257 I-27  21780 8779 KSC-1-148 P H 2-NO₂ Me H H 25 ± 11 11 ± 6 12 6229 I-29  50513 8779 KSC-1-238 P H H H Me H 27 ± 14 13 ± 1 34 6275 I-31 15992 8779 KSC-1-216 P H 2-F H Me H >30 15 ± 4 808 6255 I-32  80746 8779 KSC-1-193 P 7-Cl H H H H 70 ± 24 12 ± 4 78 6236 I-35  24546 8779 KSC-1-213 P H H CH2-* H 2-CH2-* >30 19 ± 5 28 6252 *R³ and R⁵ are connected

Formula II

TABLE 19 FORMULA II:

IC₅₀ (μM) Ub^(G76V)- GFP Cpd CID SID KU SCC # * R1 ATPase turn over II-8   2384 9225 KSC-1-289 S 2-Cl 69 ± 25 13 ± 2 230 2641 II-10 4510 9225 KSC-1-288 S 2-Br 64 ± 24 13 ± 2 8363 2640

Formula VI

TABLE 20 FORMULA VI:

IC₅₀ (μM) Ub^(G76V)- GFP turn Cpd CID SID KU SCC # * R1 ATPase over VI-16 4685 992 KSC-16-122 S 7-Br 22 ± 4 10 ± 2 0876 399 43

Formula XI

TABLE 21 Formula XI:

IC₅₀ (μM) Ub^(G76V)-GFP Cpd CID SID KU SCC # * R2 ATPase turn over XI-9 4687 3821 9931 3588 KSC-16-188 S

>30 9.2 ± 1.2

Formula XII

TABLE 22 FORMULA XII:

IC₅₀ (μM) Ub^(G76V)-GFP Cpd # CID SID KU SCC # * R4 ATPase turn over XII-1  49852 170 10422 1951 KSC-25-6  S

38 ± 7  20 ± 3  XII-2  49830 271 10390 4178 KSC-16-243 S

52 ± 10 19 ± 4  XII-9  46931 215 99437 739 KSC-16-227 S

47 ± 22 4.5 ± 0.5 XII-11 46873 814 99313 590 KSC-16-190 S

>30 8.1 ± 1.8

Formulas XXII-XXIV

TABLE 23 IC₅₀ (μM) Ub^(G76V)-GFP Cpd CID SID KU SCC # * ATPase turn over XXII 6968 40 8779 6233 KSC-1-152  P

Formula XXII 26 ± 4 7.3 ± 2 XXIII 1337 599 9209 3137 KSC-1-203  P

Formula XXIII 33 ± 8  12 ± 1 XXIV 4687 3817 9931 3595 KSC-16-197 S

Formula XXIV >30   11 ± 0.8

Formula XXV

TABLE 24 Formula XXV

IC₅₀ (μM) Ub^(G76V)- KU SCC GFP Cpd # CID SID # * R1 R2 ATPase turn over XXV-2 4685 9923 KSC-16- S 3-Cl H 34 ± 16 12 ± 2 0879 9936 163 XXV-3 4685 9923 KSC-16- S 3-Cl 4- 34 ± 14 13 ± 1 0870 9937 164 OMe

Formula XXVI-XLI

TABLE 25 IC₅₀ (μM) Ub^(G76V)-GFP Cpd # KU SCC # * ATPase turn over XXVI KSC-16-16 P

Formula XXVI ND (no data) 10.4 ± 1.5  XXVII KSC-16-22 P

Formula XXVII ND 13 ± 3  XXVIII KSC-16-11 P

Formula XXVIII ND 17 ± 3  XXIX KSC-16-14 P

Formula XXIX ND 18 ± 3  XXX KSC-16-18 P

Formula XXX ND 14 ± 3  XXXI KSC-16-45 P

Formula XXXI ND 8 ± 2 XXXII KSC-16-46 P

Formula XXXII ND 2.6 ± 0.2 XXXIII KSC-16-47 P

Formula XXXIII ND 3.4 ± 0.6 XXXIV KSC-16-48 P

Formula XXXIV ND 5.9 ± 0.8 XXXV KSC-16-49 P

Formula XXXV ND 6.1 ± 0.6 XXXVI KSC-16-50 P

Formula XXXVI ND 15 ± 2  XXXVII KSC-16-51 P

Formula XXXVII ND  13 ± 1.4 XXXVIII KSC-16-53 P

Formula XXXVIII ND  16 ± 1.4 XXXIX KSC-16-55 P

Formula XXXIX ND 6.3 ± 0.6 XL KSC-16-57 P

Formula XL ND  10 ± 1.4 XLI KSC-16-59 P

Formula XLI ND 6.5 ± 0.8

Formulas XLII and XLIII

TABLE 26 IC₅₀ (μM) Ub^(G76V)-GFP Cpd # CID SID KU SCC # * ATPase turn over XLII 46873 820 99313 589 KSC-16-189 S

Formula XLII >30 11 ± 2 XLIII 49830 263 10390 4177 KSC-16-241 S

Formula XLIII 30 ± 6 16 ± 2

COMPARATIVE EXAMPLES

In the following Tables 27-33, compounds are shown that did not decrease either p97 or Ub^(G76V)-GFP to 20 μM or less.

Formula I

TABLE 27 Formula I:

IC₅₀ (μM) Ub^(G76V)-GFP Cpd # CID SID KU SCC # * R1 R2 R3 R4 R5 ATPase turn over I-7  20472 8779 KSC-1-147 P H 4-Br H H H 72 ± 14 25 ± 10 40 6235 I-33 80800 8779 KSC-1-194 P 7-Cl 4-Me H H H 39 ± 13 24 ± 5  35 6237 I-34 80800 8779 KSC-1-195 P 7-Cl 4-OMe H H H 25 ± 11 70 ± 30 40 6238

Formula II

TABLE 28 FORMULA II:

IC₅₀ (μM) Ub^(G76V)- GFP turn Cpd # CID SID KU SSC # * R1 ATPase over II-20 46829 9923 KSC-16-150 S 3-CF3 50 ± 16 21 ± 1 335 9925

Formula VI

TABLE 29 FORMULA VI:

IC₅₀ (μM) Ub^(G76V)- GFP turn Cpd # CID SID KU SCC # * R1 ATPase over VI-8 4685 9923 KSC-16-167 S 7-CN 25 ± 10 21 ± 3 0869 9929

Formula XI

TABLE 30 Formula XI:

Formula XI IC₅₀ (μM) Ub^(G76V)-GFP Cpd # CID SID KU SCC # * R2 ATPase turn over XI-2  4985 2179 1042 2194 4 KSC-25-10  S

31 ± 7  47 ± 9  XI-3  4985 2174 1042 2194 5 KSC-25-12  S

49 ± 10 108 ± 31  XI-4  4985 2178 1042 2194 6 KSC-25-16  S

>>30 45 ± 18 XI-7  4985 2175 1042 2195 4 KSC-25-17  S

60 ± 23 37 ± 7  XI-19 4983 0268 1039 0417 0 KSC-16-272 S

 >30 >>20

Formula XII

TABLE 31 FORMULA XII:

                IC₅₀ (μM) Ub^(G76V)-GFP Cpd # CID SID KU SCC # * R4 ATPase turn over XII-3 4983 0254 10390  4179 KSC-16-251 S

33 ± 7 23 ± 2

Formula XXV

TABLE 32 Formula XXV

                    IC₅₀ (μM) Ub^(G76V)-GFP Cpd # CID SID KU SCC # * R1 R2 ATPase turn over XXV-1 1732 9923 KSC-16- S 4-OMe 4-OMe 22 ± 4  23 ± 3 4423 9924 149 XXV-4 4685 9923 KSC-16- S 3-Cl 4-Me  49 ± 19  30 ± 4 0872 9940 174 XXV-5 4685 9923 KSC-16- S 3-Cl 4-Cl 117 ± 5.8 21 ± 2 0878 9942 176 XXV-6 4685 9923 KSC-16- S 4-Me 4-Me  54 ± 19  21 ± 4 0875 9944 151

Formulas XLIV-XLXI

TABLE 33 IC₅₀ (μM) Ub^(G76V)-GFP Cpd # CID SID KU SCC # * ATPase turn over XLIV  83228 2 8779 6232 KSC-1-151 P

Formula XLIV 23 ± 6  36 ± 9  XLV  83228 3 9209 3143 KSC-1-260 P

Formula XLV 53 ± 18 31 ± 6  XLVI 29502 40 8779 6276 KSC-1-240 P

Formula XLVI 34 ± 10  36 ± 0.3 XLVII 13306 69 8779 6248 KSC-1-208 P

Formula XLVII 34 ± 11 35 ± 5  XLVIII 29556 41 8779 6277 KSC-1-241 P

Formula XLVIII 22 ± 12 39 ± 18 XLIX 34198 14 9209 3139 KSC-1-239 P

Formula XLIX 43 ± 13 70 ± 16 LI 1091 839 920 931 40 KSC-1-242 P

Formula L 57 ± 15 56 ± 10 LI 2932 797 920 9314 2 KSC-1-254 P

Formula LI 30 ± 6  34 ± 6 

Compound Derivatives

In one embodiment, a compound of the present invention is a derivative of one of the compounds disclosed herein. In another embodiment of the present invention, an inhibitor of p97 ATPase is identified by assaying any of the compound derivatives of Formulas LII through LXVI in the ATPase assay as described in Example 7.

For example, a compound derivative is represented by Formula LII:

For Formula LII above, n is 0, 1 or 2. A compound of Formula LII can be synthesized as detailed in Example 3.

In another example, a compound derivative is represented by Formula LIII:

For Formula LIII above, R¹ and R² can vary independently. R¹ and R² are independently selected from hydrogen (H), methyl, ethyl, propyl, or butyl. A compound of Formula LIII can be synthesized as detailed in Example 3.

In another example, a compound derivative is represented by Formula LIV:

For Formula LIV above, R¹ is selected from H, methyl, F, Cl, Br, and OMe on any position in the ring. R² is selected from hydrogen (H), methyl, ethyl, propyl and butyl. A compound of Formula LIV can be synthesized as detailed in Example 3.

In another example, a compound derivative is represented by Formula LV:

For Formula LV above, R¹ is selected from H, methyl, F, Cl, Br, and OMe on any position in the ring. A compound of Formula LV can be synthesized as detailed in Example 3.

In another example, a compound derivative is represented by Formula LVI:

For Formula LVI above, R¹ and R² vary independently. R¹ and R² are selected from H, methyl, F, Cl, Br, and OMe on any position in the ring. A compound of Formula LVI can be synthesized as detailed in Example 3.

In another example, a compound derivative is represented by Formula LVII:

For Formula LVII above, X is selected from O, NMe (nitrogen-methyl), NEt (nitrogen-ethyl), and NPh (nitrogen-phenyl) at any position in the ring; n is −1, 0, 1, or 2; and m is 1, 2, 3, or 4. R¹ and R² can vary independently. R¹ and R² are independently selected from H, Me, F, Cl, Br, and OMe at any position on the ring. A compound of Formula LVII can be synthesized as detailed in Example 4.

In another example, a compound derivative is represented by Formula LVIII:

For Formula LVIII above, X is selected from 0, NMe (nitrogen-methyl), NEt (nitrogen-ethyl), and NPh (nitrogen-phenyl) at any position in the ring; n is −1, 0, 1, or 2; and m is 1, 2, 3, or 4. R¹ is selected from H, Me, F, Cl, Br, and OMe on any position on the ring. A compound of Formula LVIII can be synthesized as detailed in Example 4.

In another example, a compound derivative is represented by Formula LIX:

For Formula LIX above, R¹, R² and R³ can vary independently and are each independently selected from H, A(CH₂)_(n)CH₃, and A(CH₂)_(n)X, where n is 0, 1, 2, 3, 4 or 5, A=O, S or NH and X is heteroaryl, O(alkyl), S(alkyl), (O-alkyl)₂, or (S-alkyl)₂. A compound of Formula LIX can be synthesized as detailed in Example 5.

In another example, a compound derivative is represented by Formula LX:

For Formula LX above, R¹ is selected from H, A(CH₂)_(n)CH₃, and A(CH₂)_(n)X, wherein n is 0, 1, 2, 3, 4, or 5, A is O, S or NH and X is selected from heteroaryl, O(alkyl), S(alkyl), (O-alkyl)₂, and (S-alkyl)₂. A¹ is selected from O, S, Se, N, NH, CH, CH₂, CHalkyl, and Calkyl; and A² is selected from N, NH, CH, and Calkyl. A compound of Formula LVX can be synthesized as detailed in Example 5.

In another example, a compound derivative is represented by Formula LXI:

For Formula LXI above, R¹, R² and R³ can vary independently. R¹, R² and R³ are independently selected from alkyl, alkoxyalkyl, and aminoalkyl. R⁴ is selected from H, halogen, alkyl, and alkyoxy. X is selected from CH₂, O NH, NMe, NEt, NCOMe, and NPh. Y is selected from NH, O, S, [H, H], and n is 0 or 1. A compound of Formula LXI can be synthesized as detailed in Example 6.

In another example, a compound derivative is represented by Formula LXII:

For Formula LXII above, n is −1, 0, 1, or 2; m is 0, 1, or 2, and X is selected from CH₂, O, NMe, NEt, and NPh at any position in the ring. A compound of Formula LXII can be synthesized following a scheme as shown in Example 4.

In another example, a compound derivative is represented by Formula LXIII:

For Formula LXIII above, R¹ and R² vary independently. R¹ and R² independently selected from H, Me, Et, Pr, and Bu. n is −1, 0, 1, or 2. X is selected from CH₂, O, NMe, NEt, NPh at any position in the ring. A compound of Formula LXIII can be synthesized following a scheme as shown in Example 4.

In another example, a compound derivative is represented by Formula LXIV:

For Formula LXIV, R¹ is selected from H, Me, F, Cl, Br, and OMe at any position on the ring. R² is selected from H, Me, Et, Pr, and Bu. X is selected from CH₂, O, NMe, NEt, and NPh at any position on the ring. n is −1, 0, 1, or 2. A compound of Formula LXIV can be synthesized following a scheme as shown in Example 4.

In another example, a compound derivative is represented by Formula LXV:

For Formula LXV, R¹ is selected from H, Me, F, Cl, Br, and OMe at any position on the ring. R² is selected from H, Me, Et, Pr, and Bu. X is selected from CH₂, O, NMe, NEt, and NPh at any position on the ring. n is −1, 0, 1, or 2. A compound of Formula LXV can be synthesized following a scheme as shown in Example 4.

In another example, a compound derivative is represented by Formula LXVI:

For Formula LXVI, R¹ and R² can vary independently. R¹ and R² are independently selected from H, Me, F, Cl, Br, and OMe at any position on the ring. X is selected from CH₂, O, NMe, NEt, and NPh at any position on the ring. n is −1, 0, 1, or 2. A compound of Formula LXVI can be synthesized following a scheme as shown in Example 4.

Isomers

Compounds of this invention may contain asymmetrically substituted carbon atoms in the R or S configuration, wherein the terms “R” and “S” are as defined in Pure Appl. Chem. (1976) 45, 11-30. Compounds having asymmetrically substituted carbon atoms with equal amounts of R and S configurations are racemic at those atoms. Atoms having excess of one configuration over the other are assigned the configuration in excess, preferably an excess of about 85%-90%, more preferably an excess of about 95%-99%, and still more preferably an excess greater than about 99%. Accordingly, this invention is meant to embrace racemic mixtures and relative and absolute diastereoisomers of the compounds thereof.

Compounds of this invention may also contain carbon-carbon double bonds or carbon-nitrogen double bonds in the Z or E configuration, in which the term “Z” represents the larger two substituents on the same side of a carbon-carbon or carbon-nitrogen double bond and the term “E” represents the larger two substituents on opposite sides of a carbon-carbon or carbon-nitrogen double bond. The compounds of this invention may also exist as a mixture of “Z” and “E” isomers.

Compounds of this invention may also exist as tautomers or equilibrium mixtures thereof wherein a proton of a compound shifts from one atom to another. Examples of tautomers include, but are not limited to, keto-enol, phenol-keto, oxime-nitroso, nitro-aci, imine-enamine and the like.

In one embodiment, isomers of the disclosed compounds are regioisomers or stereoisomers.

Compound Analogs

The compounds disclosed herein may be further modified to enhance solubility, detection and/or delivery in the body. The following modifications are not necessarily exclusive to another and can be combined. For example, a PEGylated and fluorescently labeled compound is disclosed.

In one embodiment, a compound of the present invention is fluorescently labeled. Suitable fluorescent labels are well known. A fluorescent label to be added to an inhibitor compound includes, but is not limited to, NBD-Cl (4-Chloro-7-nitro-2,1,3-benzoxadiazole), R—NCO (isocyanate), R—NCS (FITC).

In one embodiment, a compound of the present invention is biotinylated. Biotinylation is carried out using a biotin derivative. Examples of biotin derivatives include ester-biotin, amine-biotin, amide-biotin, and OH-biotin.

In one embodiment, a compound of the present invention is PEGylated with at least one PEG moiety. It is well known in the art that polyalkylene glycols, such as polyethylene glycol (PEG), may be attached to a therapeutic agent. Polyalkylene glycolated (PAGylated) therapeutic agents, and in particular, PEGylated therapeutic agents, have been reported to increase solubility, circulating life, safety, decrease renal excretion, and decrease immunogenicity thus potentially providing a method of improved drug delivery. A PEGylated therapeutic agent may exhibit (a) increased plasma circulatory half lives in vivo compared to the corresponding non-PEGylated compound, (b) enhanced therapeutic indices compared to the corresponding non-PEGylated compounds and (c) increased solubility compared to the corresponding non-PEGylated compounds, effecting possible improved drug delivery. Examples in which PEGylation has been used to effect drug delivery are disclosed, for example, in U.S. Pat. Nos. 6,623,729, 6,517,824, 6,515,017, 6,217,869, 6,191,105, 5,681,811, 5,455,027, U.S. Published Patent Application Nos. 20040018960, 20030229010, 20030229006, 20030186869, 20030026764, and 20030017131 U.S. Pat. No. 6,214,966, U.S. Published Patent Application No 2003000447, and U.S. Published Patent Application No. 2001021763 describe soluble, degradable poly(ethylene glycol) derivatives for controlled release of bound molecules into solution. Recent reviews on PEGylation are provided in, for example, Greenwald R. B., Choe Y. H., McGuire J., Conover C D. Adv. Drug Del. Rev. 2003, 55, 217, Molineux G. Pharmacotherapy 2003, (8 Pt 2), 3S-8S, Roberts M. J., Bentley, M. D., Harris J. M. Adv. Drug Deliv. Rev. 2002, 54, 459, Bhadra D., Bhadra S., Jain P., Jain N. K. Pharmazie 2002, 57, 5, Greenwald R B. J. Controlled Release 2001, 74, 159, Veronese F. M., Morpurgo M. Farmaco. 1999, 54, 497 and Zalipsky S. Adv. Drug Deliv. Rev. 1995, 76, 157. In particular, the compounds of formulas I through LXVI as described herein, may be PEGylated.

Pharmaceutical Salts

The phrase “pharmaceutically acceptable” is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of the medical arts, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Compounds of the present invention, including all analogs and isomers of the compounds may exist as acid addition salts, basic addition salts or zwitterions. Salts of compounds disclosed herein are prepared during their isolation or following their purification. Acid addition salts are those derived from the reaction of a compound of the present invention with acid. Accordingly, salts including the acetate, adipate, alginate, bicarbonate, citrate, aspartate, benzoate, benzenesulfonate (besylate), bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, formate, fumarate, glycerophosphate, glutamate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactobionate, lactate, maleate, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, phosphate, picrate, propionate, succinate, tartrate, thiocyanate, trichloroacetic, trifluoroacetic, para-toluenesulfonate and undecanoate salts of the compounds having any of formulas I through LXVI are meant to be embraced by this invention. Basic addition salts of compounds are those derived from the reaction of the compounds with the bicarbonate, carbonate, hydroxide or phosphate of cations such as lithium, sodium, potassium, calcium and magnesium. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and in the Journal of Pharmaceutical Science, 66, 2 (1977), the disclosures of each of which are hereby incorporated by reference.

Pharmaceutical Formulations and Dosage Forms

When employed as pharmaceuticals, the compounds of any of Formulas (I-LXVI) can be administered in the form of pharmaceutical compositions. These compositions can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal, and can be prepared in a manner well known in the pharmaceutical art.

This invention also includes pharmaceutical compositions which contain, as the active ingredient, one or more of the compounds of Formulas I-LXVI (including analogs and isomers thereof) above in combination with one or more pharmaceutically acceptable carriers. In making the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

In preparing a formulation, the active compound can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

The active compound can be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in can be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face masks tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices which deliver the formulation in an appropriate manner.

The amount of compound or composition administered to a patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration, and the like. In therapeutic applications, compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is referred to as “therapeutically effective amount.” Effective doses will depend on the disease condition being treated as well as by the judgement of the attending clinician depending upon factors such as the severity of the disease, the age, weight and general condition of the patient, and the like.

The compositions administered to a patient can be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques, or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparations typically will be between 3 and 11, more preferably from 5 to 9 and most preferably from 7 to 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of pharmaceutical salts.

Compounds disclosed herein may be administered, for example, bucally, ophthalmically, orally, osmotically, parenterally (intramuscularly, intraperintoneally intrasternally, intravenously, subcutaneously), rectally, topically, transdermally, vaginally and intraarterially as well as by intraarticular injection, infusion, and placement in the body, such as, for example, the vasculature by means of, for example, a stent.

The present invention also includes pharmaceutical kits useful, for example, in the treatment of cancers, which comprise one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (I-LXVI). Such kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.

EXAMPLES Example 1 Purchased Compounds

Compounds shown in Tables 1-33 were either synthesized or purchased as indicated in the Tables, wherein S indicates synthesized and P indicates purchased. Table 34 below provides company information for purchased compounds.

TABLE 34 Cpd # KU SCC # Company I-1 KSC-1-150 Chembridge I-2 KSC-1-145 Aldrich I-3 KSC-1-146 Chembridge I-4 KSC-1-149 Chembridge I-5 KSC-1-226 Chemdiv I-6 KSC-1-236 Princeton Biomecular Research, Inc I-7 KSC-1-147 Chembridge I-8 KSC-1-234 Princeton Biomecular Research, Inc I-9 KSC-1-227 Chemdiv I-10 KSC-1-224 Chemdiv I-11 KSC-1-200 Ryan Scientific, Inc. I-12 KSC-1-211 Ryan Scientific I-13 KSC-1-212 Ryan Scientific I-14 KSC-1-214 Ryan Scientific I-15 KSC-1-215 Ryan Scientific I-16 KSC-1-217 Chemdiv I-17 KSC-1-219 Chemdiv I-20 KSC-1-220 Chemdiv I-21 KSC-1-221 Chemdiv I-22 KSC-1-222 Chemdiv I-23 KSC-1-223 Chemdiv I-24 KSC-1-225 Chemdiv I-25 KSC-1-258 Interchim I-26 KSC-1-218 Chemdiv I-27 KSC-1-148 Chembridge I-28 KSC-1-235 Princeton Biomecular Research, Inc I-29 KSC-1-238 Princeton Biomecular Research, Inc I-30 KSC-1-237 Princeton Biomecular Research, Inc I-31 KSC-1-216 Chemdiv I-32 KSC-1-193 Albany Molecular Research, Inc. I-33 KSC-1-194 Albany Molecular Research I-34 KSC-1-195 Albany Molecular Research I-35 KSC-1-213 Ryan Scientific XXII KSC-1-152 Chembridge XLIV KSC-1-151 Chembridge XLV KSC-1-260 interchim XLVI KSC-1-240 Princeton Biomecular Research, Inc XLVII KSC-1-208 Ryan Scientific XLVIII KSC-1-241 Princeton Biomecular Research, Inc XXIII KSC-1-203 Ryan Scientific XLIX KSC-1-239 Princeton Biomecular Research L KSC-1-242 Princeton Biomecular Research LI KSC-1-254 Princeton Biomecular Research, Inc II-1 KSC-1-153 Chembridge II-2 KSC-1-251 Princeton Biomecular Research II-3 KSC-1-246 Princeton Biomecular Research II-4 KSC-1-249 Princeton Biomecular Research II-5 KSC-1-245 Princeton Biomecular Research II-6 KSC-1-250 Princeton Biomecular Research II-7 KSC-1-252 Princeton Biomecular Research II-9 KSC-1-247 Princeton Biomecular Research II-11 KSC-1-248 Princeton Biomecular Research II-12 KSC-1-253 Princeton Biomecular Research II-13 KSC-1-244 Princeton Biomecular Research II-17 KSC-1-259 interchim

Example 2 Compound Synthesis

In general, compounds of the invention, including salts and solvates thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of numerous possible synthetic routes. The reactions for preparing compounds of the invention can be carried out in suitable solvents which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, i.e., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected.

Preparation of compounds of the invention can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd. Ed., Wiley & Sons, Inc., New York (1999), which is incorporated herein by reference in its entirety.

Reactions can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

Synthesized compounds were synthesized using one of the following three synthesis schemes. Synthesis scheme I was carried out with reference to Gavish et al. WO 2008/023357A1.

Synthesis scheme II was carried out with reference to Gahman et al. US 2009/0209536 A1.

Representative compound synthesis: N2,N4-dibenzyl-8-methoxyquinazoline-2,4-diamine. (Compound #VI-5). To a suspension of 2,4-dichloro-8-methoxyquinazoline (50 mg, 0.22 mmol) in acetonitrile (1 mL) was added benzylamine (0.12 mL, 1.09 mmol, 5 equiv.). The mixture was heated to 180° C. for 1 h under microwave irradiation. The concentrated residue was purified by silica gel chromatography (Ethyl acetate) to give a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.44-7.18 (m, 11H), 7.11 (dd, J=1.6, 7.9 Hz, 1H), 7.07-6.93 (m, 2H), 5.86 (s, br. 1H), 5.49 (s, br. 1H), 4.78 (d, J=5.7 Hz, 2H), 4.75 (d, J=5.7 Hz, 2H), 3.99 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 160.1, 159.3, 153.2, 144.2, 140.2, 138.7, 128.7, 128.4, 128.0, 127.5, 126.8, 120.4, 112.5, 111.2, 110.9, 55.9, 45.7, 45.2. HRMS (m/z): calcd for C₂₃H₂₃N₄O (M+H) 371.1872; found 371.1871.

Some specific examples of synthesis schemes are shown as follows.

Specifically, for XXIV, the synthesis scheme is as shown below:

A synthesis scheme for Formula II is shown below:

A synthesis scheme for Formula III is shown below:

A synthesis scheme for Formula IV is shown below:

A synthesis scheme for Formula V is shown below:

A synthesis scheme for Formula VI is shown below:

Example 3 Synthesis of Compound Derivatives LII, LIII, LIV, LV and LVI

Synthesis Schemes with reference to Kohn et al., 1983, J. Am. Chem. Soc, 105, 4106-4108.

Example 4 Synthesis of Compound Derivatives LVII, LVIII, LXII, LXIII, LXIV, LXV, and LXVI

Synthesis scheme for LVII. For LVIII and LXII-LXVI, reference is made to the scheme below, with reference to schemes disclosed herein and Zaugg, 1984, Synthesis, 86-110.

Example 5 Synthesis of Compound Derivatives LIX and LXI

Synthesis scheme for LIX with reference to Tikad et al., 2006, Synlett, 1938-1942.

Synthesis scheme for LX with reference to above reaction and reference for LIX.

Example 6 Synthesis of Compound Derivative LXI

Synthesis scheme for LXI with reference to Zaugg, 1984, Synthesis, 86-110.

Example 7 ATPase Assay

Assay Buffer (20 μL of 2.5× concentration, where 1×=50 mM Tris pH 7.4, 20 mM MgCl₂, 1 mM EDTA, 0.5 mM TCEP) was dispensed into each well of a 96 well plate. Purified p97 (25 μL of 50 μM) was diluted in 975 μL, of 1× Assay Buffer and 10 μL was dispensed in each well. Test compound (10 μL) or 5% DMSO (10 μL) was then added to each well and the plate was incubated at room temperature for 10 min. The ATPase assay was carried out by adding to each well 10 μL of 500 μM ATP (pH 7.5), incubating at room temperature for 60 min, and then adding 50 μL Kinase Glo Plus reagent (Promega) followed by a final 10 min incubation at room temperature in the dark. Luminescence was read on an Analyst AD (Molecular Devices). Compounds were assayed at a range of concentrations (0, 0.048, 0.24, 1.2, 6, 30 μM) in triplicate. The percent of remaining activity for each reaction was calculated using the following mathematical expression: ((Test Compound−High Control)/(Low Control−High Control))*100. Test_Compound is defined as wells containing test compound, Low_Control is defined as wells containing DMSO, High_Control is defined as wells containing no p97 protein. IC₅₀ values were calculated from fitting the percentage of remaining activity (% RA) with various concentrations of compounds to a Langmuir equation [% RA=100/(1+[Compound]/IC₅₀)] by non-linear regression analysis using the JUMP IN program. The result was expressed as mean +/− standard error. For assays with Myriad 12 and 19, 100 μL of biomol green reagent (Enzo Life Sciences) was added to each well instead of kinase Glo Plus (Promega) and absorbance at 630 nm was measured. This was done because these compounds interfered with luciferase activity. For assays with compounds that interfered with luciferase activity, 100 μL of biomol green reagent (Enzo Life Sciences) was added to each well instead of kinase Glo Plus (Promega) and absorbance at 630 nm was measured.

Example 8 Ub^(G76V)-GFP Degradation Assay

Two GFP images with 100 ms exposure time per well were acquired and the average GFP intensity per area of a HeLa cell was determined by using MetaXpress software. Mean GFP intensity of 300-500 cells was calculated using Excel. Normalized GFP intensity was calculated using the following formula: (Test compound−Background)/(Basal GFP intensity—Background). Where: Test compound is defined as Mean GFP intensity of Ub^(G76V)-GFP-expressing cells treated with the test compound. Background is defined as background GFP intensity of HeLa cells. Basal GFP intensity is defined as mean GFP intensity of Ub^(G76V)-GFP-expressing cells treated with DMSO. The degradation rate constant (k) was obtained from the slope of the linear range of plotting Ln (Normalized GFP intensity) versus time ranging from 90 to 180 min. The percent of remaining k for each compound is calculated using the following formula: (Test compound/DMSO control)*100. Where: Test_compound is defined as k determined from wells containing test compound, DMSO control is defined as k determined from wells containing DMSO. IC₅₀ values were calculated from fitting the percentage of remaining k (% k) with various concentrations of compounds to a Langmuir equation [% k=100/(1+[Compound]/IC₅₀)] by non-linear regression analysis using the JUMP IN program. The result was expressed as mean +/− standard error.

Example 9 NMR Analysis of Compounds

NMR data for all compounds in attached in the Appendix.

¹H and ¹³C spectra were recorded on a Bruker Avance 400 or 500 MHz spectrometer. Chemical shifts are reported in parts per million and were referenced to residual proton solvent signals. 

What is claimed is:
 1. A composition suitable for decreasing p97 ATPase activity and/or degradation of a p97 dependent ubiquitin-proteasome substrate, comprising: a compound of Formula VII, IX, XII, XX, XXI or XLIII, or a pegylated analog of the compound, a pharmaceutically acceptable salt of the compound or the analog, or any regioisomer or stereoisomer of the compound:

Wherein, for Formula VII, R¹, n, X, and Y are selected from the group consisting of combinations listed in Table 7: TABLE 7 Cpd # R1 n X Y VII-3 H 1 O H, H VII-4 H 1 NH H, H VII-5 8-OMe 1 NH H, H VII-6 8-OMe 1 O H, H VII-7 8-OH 1 O H, H VII-8 8-Ph 1 O H, H VII-9 8-OCH₂CH₂OH 1 O H, H VII-10 8OCH₂CH₂NEt₂ 1 O H, H VII-11 8-p-OMePh 1 O H, H VII-12 8-OMe 1 NMe H, H VII-13 8-OMe 1 NCOMe H, H VII-14 8-OCH₂CH₂OMe 1 O H, H VII-16 8-OMe 0 NH NH VII-17 8-OMe 0 O Y,Y together form one O as oxygen of carbonyl incorporating both Y's VII-15 8-n-Butyl 1 O H, H

Wherein, for Formula XII, R⁴ is selected from the group consisting of the moieties listed in Table 10: TABLE 10.1 Cpd # R4 Cpd # R4 XII-4

XII-8

XII-7

XII-10

XII-2

XII-11

XII-9

Wherein for Formula XXI, R¹ is 5,6-dimethyl;


2. A composition of claim 1, further comprising a pharmaceutically acceptable carrier.
 3. A composition of claim 1, wherein the isomer is a regioisomer or stereoisomer.
 4. A composition of claim 1, wherein the compound is represented by Formula VII or XXI.
 5. A composition of claim 4 wherein the compound is represented by Formula VII.
 6. A composition suitable for decreasing p97 ATPase activity and/or degradation of a p97 dependent ubiquitin-proteasome substrate, comprising: a compound of Formula LII, LVII, LVIII, LIX, LX, LXI, LXII or a pegylated analog of the compound, a pharmaceutically acceptable salt of the compound or the analog, or any regioisomer or stereoisomer of the compound:

Formula LII wherein N is 0, 1 or 2; Formula LVII wherein X is oxygen or N—R′ wherein R′ is methyl, ethyl or phenyl; n is −1, 0, 1 or 2; m is 1, 2, 3 or 4; R¹ and R² are independently selected from the group consisting of hydrogen, methyl, fluoro, Chloro, bromo and methoxy;

Formula LVIII wherein X is O, NR′ wherein Formula LIX wherein R¹, R², R³ are R′ is methyl, ethyl or phenyl; n is −1, 0, 1 or 2; independently selected from the group m is 1, 2, 3 or 4; R¹ is hydrogen, methyl fluoro, consisting of hydrogen, A(CH₂)_(n)CH₃ and chloro, bromo or methoxy A(CH₂)_(n)X wherein n is 0, 1, 2, 3, 4 or 5; A is O, S or NH; and X is heteroaryl, O-alkyl, S- alkyl, (O-alkyl)₂ or (S-alkyl)₂

Formula LX wherein, A¹ is O, S, Se, N, NH, Formula LXI wherein R¹, R², R³ are CH, CH₂, CHalkyl, or Calkyl; n is 1 or 2; A² is independently selected from the group N, NH, CH, or Calkyl; and R¹ is selected from consisting of alkyl, alkoxyalkyl and the group consisting of H, A(CH₂)_(m)CH₃, and aminoalkyl A(CH₂)_(n)X, wherein A is O, S or NH and X is heteroaryl, O(alkyl), S(alkyl), (O-alkyl)₂, or (S- alkyl)₂; and m is 0, 1, 2, 3, 4, or 5

Formula LXII wherein n is −1, 0, 1 or 2; m is 0, 1 or 2; and X is CH₂, O or NR′; and R′ is methyl, ethyl or phenyl.


7. A composition of claim 6, further comprising a pharmaceutically acceptable carrier.
 8. A composition of claim 6, wherein the isomer is a regioisomer or stereoisomer.
 9. A composition of claim 6, wherein the compound is represented by Formula LII, LVII, LVIII or LXII.
 10. A composition of claim 6 wherein the compound is represented by Formula LVII, LVIII or LXII.
 11. A composition of claim 6 wherein the compound has formula LIX-A


12. A composition suitable for decreasing p97 ATPase activity and/or degradation of a p97 dependent ubiquitin-proteasome substrate, comprising: a compound of Formula VIII or a pegylated analog of the compound, a pharmaceutically acceptable salt of the compound or the analog, or any regioisomer or stereoisomer of the compound:


13. A composition of claim 1 wherein the compound of Formula VII, IX, XII, XX, XXI or XLIII is labeled with a fluorescent label selected from the group consisting of 4-chloro-7-nitor-2,1,3-benzoxadiaxole, R—NCO wherein the R group is a stable organic fluorescent group and R—NCS wherein the R group is a stable organic fluorescent group.
 14. A composition of claim 1 wherein the compound of Formula VII, IX, XII, XX, XXI or XLIII is biotinylated with a biotin derivative selected from the group consisting of ester-biotin, amin-biotin, amide-biotin and hydroxyl-biotin.
 15. A composition of claim 6 wherein the compound of Formula LII, LVII, LVIII, LIX, LX, LXI, LXII is labeled with a fluorescent label selected from the group consisting of 4-chloro-7-nitor-2,1,3-benzoxadiaxole, R—NCO wherein the R group is a stable organic fluorescent group and R—NCS wherein the R group is a stable organic fluorescent group.
 16. A composition of claim 6 wherein the compound of Formula LII, LVII, LVIII, LIX, LX, LXI, LXII is biotinylated with a biotin derivative selected from the group consisting of ester-biotin, amine-biotin, amide-biotin and hydroxyl-biotin.
 17. A composition of claim 12 wherein the compound of Formula VIII is labeled with a fluorescent label selected from the group consisting of 4-chloro-7-nitor-2,1,3-benzoxadiaxole, R—NCO wherein the R group is a stable organic fluorescent group and R—NCS wherein the R group is a stable organic fluorescent group.
 18. A composition of claim 12 wherein the compound of Formula VIII is biotinylated with a biotin derivative selected from the group consisting of ester-biotin, amin-biotin, amide-biotin and hydroxyl-biotin. 